1. Volume 89, Issue 4, Pages 597-606 (May 1997)
mRNA Silencing in Erythroid Differentiation: hnRNP K and hnRNP E1 Regulate 15- Lipoxygenase Translation from the 3′ End 
Dirk H Ostareck, Antje Ostareck-Lederer, Matthias Wilm, Bernd J Thiele, Matthias Mann, Matthias W Hentze 
Cell 
Volume 89, Issue 4, Pages (May 1997)
DOI: /S (00)80241-X

2. Figure 1
Identification of the LOX Regulatory Proteins as hnRNPs K and E1
Scheme of the LOX mRNA with its 3′ UTR–regulatory (10R) and nonregulatory region (NR).
(A) Silver-stained gel with final wash ([w], lanes 1 and 3) and eluates ([e], lanes 2 and 4) after protein purification using NR (lanes 1 and 2) or 10R (lanes 3 and 4) affinity resins. Nonspecific peptides are labeled (n.s.); three bands of 66 kDa, 48 kDa, and 43 kDa specifically elute from 10R.
(B) Nanoelectrospray mass spectrum of the 43 kDa band ([A], lane 4); see Experimental Procedures.
(C) Parent ion scan: detection of ions leading to Ile/Leu–containing peptides.
(D) A sequence tag of a peptide ion identified in (C) was assembled in the high m/z part of the spectrum. Arrows indicate the sequence for peptide sequence tag searching. An alignment of the complete C-terminal ion series is shown.
Cell  , DOI: ( /S (00)80241-X)

3. Figure 2
HnRNP E1 and hnRNP K Specifically Regulate LOX mRNA Translation in Rabbit Reticulocyte Lysate
(A and B) LOX-10R ([A], lanes 1–12) or LOX-NR mRNAs ([B], lanes 1–12) were cotranslated with CAT mRNA as internal control. In (A) and (B), dialysis buffer (lanes 1 and 12) or protein fractions purified from NTA or DICE columns were added as indicated, hnRNPs E1/K in a 1:3 ratio. [35S]Met incorporation into translation products was determined by phosphoimaging (Compaq Phosphor Imager with Molecular Dynamics Image Quant software), and the signals in lanes 1 and 12 were taken as 100% standards and averaged. The percentage of LOX expression normalized for the CAT internal control is shown below each lane.
(C) A 32P-labeled 10R probe was photocrosslinked to the regulatory proteins or to 150 μg of rabbit reticulocyte lysate (S100) (lane 1) at 254 nm. Note that the N-terminal histidine tag adds ∼3 kDa to the recombinant proteins.
Cell  , DOI: ( /S (00)80241-X)

4. Figure 5
HnRNPs K and E1 Silence LOX mRNA Translation in Wheat Germ Extract
LOX mRNAs bearing either two repetitive elements (LOX-2R, lanes 1–7), a mutated version of the two repeat motif (LOX-2Rmut, lanes 8–14), or the α-globin 3′ UTR (LOX-α-globin, lanes 15–20) were cotranslated with CAT mRNA (lanes 1–20) as an internal control. Dialysis buffer (lanes 1, 8, and 15); 50 ng of DICE column–purified recombinant proteins hnRNP E1, hnRNP K, hnRNP E1/K (1:3 ratio); DICE column–purified rabbit reticulocyte regulatory proteins; or recombinant proteins hnRNP A1 and IRP-1 were added as indicated. The data were quantitatively evaluated as described in the legend to Figure 2.
Cell  , DOI: ( /S (00)80241-X)

5. Figure 6 HnRNPs K and E1 Specifically Inhibit 80S Ribosome Assembly
32P-labeled RNAs were preincubated with dialysis buffer (open circles) or 500 ng of recombinant NTA-agarose purified hnRNP E1/K (1:3 ratio) (plus signs). Translation initiation complexes were subsequently allowed to assemble on the labeled LOX-2R (left panel) or LOX-2Rmut (right panel) mRNAs in cycloheximide-treated rabbit reticulocyte lysate, and resolved by centrifugation in 5%–25% linear sucrose gradients. After fractionation (lower panels), the labeled mRNAs were extracted and analyzed by autoradiography after formaldehyde/agarose gel electrophoresis (even-numbered fractions), or the radioactivity counted in the odd-numbered fractions, expressed as the percentage of total counts recovered, and plotted against the fraction numbers. The dashed line denotes the A254 absorption profile, which was identical for gradients with added buffer or recombinant proteins.
Cell  , DOI: ( /S (00)80241-X)

6. Figure 3 Translational Silencing by hnRNPs K and E1 In Vivo
(A–D) HeLa cells were transiently transfected with 5 μg of LUC reporter constructs bearing a 38 nt DICE [LUC-2R] (lanes 1–6) or a mutated version as a negative control [LUC-2Rmut] (lanes 7–12), and 5 μg of a human growth hormone (hGH) internal specificity control plasmid driven by the same promoter as the LUC reporter constructs (lanes 1–12). In addition, 10 μg of U1A (lanes 1 and 7), 5 μg (lanes 2 and 8) or 10 μg (lanes 3 and 9) of hnRNP E1; 5 μg (lanes 4 and 10) or 10 μg (lanes 5 and 11) of hnRNP K; or 5 μg of hnRNP E1 plus 5 μg of hnRNP K (lanes 6 and 12) expression plasmids were cotransfected. Lane 13 shows an analysis of untransfected cells. Parallel dishes were transfected with the same precipitate, and used for metabolic labeling for 2 hours with [35S]-Met or for preparation of total RNA and cell extract. Labeled extracts were used for immunoprecipitations with polyclonal antibodies against luciferase (A), hGH (C), and ferritin (D). Unlabeled extracts (50 ng of protein) were used for enzymatic analysis of luciferase activity (B). Luciferase activities in lanes 1 and 7, respectively, were set at 100%. (E) Northern blot of the total RNA hybridized with a luciferase probe. Equal signal intensities were also obtained with a ferritin H chain probe (for lanes 1–13) and an hGH probe (for lanes 1–12, data not shown).
Cell  , DOI: ( /S (00)80241-X)

7. Figure 4 In Vivo Association of hnRNP K and LOX mRNA
mRNPs from rabbit reticulocytes were precipitated with hnRNP K polyclonal antiserum (lanes 4 and 5), preimmune serum (lanes 2 and 3) or buffer (lanes 6 and 7). RNA was extracted from mRNPs (lane 1), or supernatants (s) and pellets (p) after immunoprecipitation, and analyzed by Northern blotting with 32P-dCTP-labeled LOX and β-glo- bin cDNAs. The signals were quantitated by phosphoimaging and expressed as supernatant-to-pellet ratios.
Cell  , DOI: ( /S (00)80241-X)

8. Figure 7
HnRNPs K and E1 Specifically Silence Both Cap-Dependent and IRES-Mediated Translation
LOX-10R mRNA as an internal positive control (lanes 1–16) was cotranslated with the following CAT reporter mRNAs (schematically depicted at the top): CAT (lanes 1–4), CAT-10R (lanes 5–8), IRES-CAT (lanes 9–12), or IRES-CAT-10R (lanes 13–16). Dialysis buffer (lanes 1, 5, 9, and 13), 3.3 mM m7GpppG cap-analog (lanes 2, 6, 10, and 14), 500 ng of NTA-agarose-purified hnRNPs E1/K (1:3 ratio, lanes 3, 7, 11, and 15), or 50 ng of DICE-purified rabbit reticulocyte regulatory proteins (lanes 4, 8, 12, and 16) were added to the translation reactions in rabbit reticulocyte lysate. The positions of 35S-methionine-labeled LOX, CAT, and IRES-CAT fusion polypeptides are indicated. Translation of LOX and CAT mRNAs was quantitated by phosphoimaging and expressed as percentages of the signal in lanes without the addition of regulatory proteins.
Cell  , DOI: ( /S (00)80241-X)